Pseudomonas treatment composition and method

ABSTRACT

A composition and method for treating or preventing infection by  Pseudomonas aeruginosa  is disclosed. The composition includes a  P. aeruginosa  pilin peptide modified to prevent oligomerization of the pilin. The method involves administered the composition to a person infected with Pseudomonas are at risk of such infection.

[0001] This application claims priority to U.S. Provisional ApplicationSer. No. 60/089,155 filed Jun. 12, 1998, which is hereby incorporated byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to a novel composition and methodfor treatment and prevention of infection by Pseudomonas aeruginosa.

REFERENCES

[0003] 1. Irvin, R. T. (1993) “Attachment and colonization ofPseudomonas aeruginosa: Role of the surface structures”, in Pseudomonasaeruginosa as an Opportunistic Pathogen, (Campa, M., M. Bendinelli, andH. Friedman, eds.), pp 1942, Plenum Press, New York.

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[0015] 13. Paranchych, W., et al. (1990) “Expression, processing, andassembly of Pseudomonas aeruginosa N-methylphenylalanine pilin”, inPseudomonas: Biotransformations, Pathogenesis and EvolvingBiotechnology, (Sliver, S., et al., eds.), pp 343-351, American Societyfor Microbiology, Washington, D.C.

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BACKGROUND OF THE INVENTION

[0044]Pseudomonas aeruginosa is a significant opportunistic pathogenthat causes a variety of life-threatening infections in immunosuppressedor immunocompromised patients [1-4]. Individuals who are at risk ofdeveloping P. aeruginosa infections include cystic fibrosis patients,bum patients, severe neutropenic patients (e.g., cancer patientsreceiving chemotherapy) and intensive care unit patients receivingrespiratory support. The cost of these infections is high, >60,000 livesper year in North America and about $5 billion/year in health carecosts.

[0045] The first step in the Pseudomonas infection process appears to bethe attachment to the host cell. This attachment is mediated by pili onthe surface of the bacterium [2, 5, 6]. P. aeruginosa uses severaladhesins to mediate attachment to mucosal surfaces, but analysis of thebinding properties of the adhesins [1, 7, 8] and binding competitionstudies [9] indicate that the pilus is the dominant adhesin responsiblefor initiating infections [1].

[0046]P. aeruginosa pili have a structure resembling a hollow tube ofabout 5.2 nm in outer diameter, 1.2 nm in central channel diameter, andan average length of 2.5 μm [10-12]. The pilus of P. aeruginosa iscomposed of multiple copies of a 13-17 kDa monomeric protein subunitcalled pilin, which are capable of self-assembling into pili.

[0047] The C-terminal region of the pilin monomer contains theepithelial cell binding domain [5, 12], and is semiconserved in sevendifferent strains of this bacterium [13, 14]. This semiconserved regionhas also been shown to bind to a minimal structural carbohydratereceptor sequence, β-GalNAc(1-4)βGal, found in glycosphingolipids,specifically asialo-GM1 and asialo-GM2 [15, 16]. There is evidence thatpili binding to a host cell is mediated multivalent binding ofC-terminal binding domains in each pili to epithelial-cell receptors,with such binding serving to mobilize receptors on the cells. This, inturn, may be responsible to cytokine, e.g., IL-8 production by the hostcells and consequent inflammatory response.

[0048] The C-terminal disulfide-bridged 17-residue region of the PAKpilin is known to be important in raising antibodies that block bindingof both bacteria or their pili to epithelial cells [6, 17, 18]. Bothmonoclonal antisera generated from P. aeruginosa pili or polyclonalantisera generated from synthetic peptides representing the receptorbinding domain of the pathogen have been shown to be efficacious inpreventing infection [19].

[0049] The ability of antibodies produced against the C-terminalpilin-peptide domain to effectively inhibit Pseudomonas infection hasbeen demonstrated (see, for example, U.S. Pat. No. 5,468,484), and theuse of the pilin-peptide domain for use in vaccination againstPseudomonas infection has also been demonstrated, e.g., U.S. Patent Nos.5,445,818, 5,494,672, and 5,612,036.

[0050] It would also be desirable to directly treat an existingPseudomonas infection, or to treat an individual at risk of Pseudomonasinfection prophylactically. Although intact pili have been proposed forthis purpose, this method is limited by the fact that isolated,self-assembled pili have the ability to provoke a strong inflammatoryresponse. Alternatively, the C-terminal pilin peptide has been proposedfor this purpose, but this approach is limited by the relatively weakbinding of the peptide to the host-cell receptor sites.

SUMMARY OF THE INVENTION

[0051] The invention includes, in one aspect, a composition for use intreating or preventing infection by Pseudomonas aeruginosa. Thecomposition comprises a P. aeruginosa pilin protein having an N-terminalpeptide region modified to prevent self assembly of the peptide. Thepeptide may be formulated in a pharmaceutically acceptable carrier, suchas an aerosolizable liquid or particle vehicle, or an injectablesolution.

[0052] In one general embodiment, the modified N-terminal peptide regionlacks an N-terminal portion of native P. aeruginosa, preferably thefirst 15 up to the first 40 amino acids residues of native P.aeruginosa, more preferably the first 25 up to the first 30 amino acids.

[0053] In another general embodiment, the N-terminal region is modified,e.g., by amino acid substitutions, to prevent or inhibit alpha-helixformation in the N-terminal region, thereby preventing the pilin peptidefrom self-assembling.

[0054] In still another embodiment, the N-terminal region of the pilinpeptide is replaced by a peptide moiety capable of forming a coiled-coilheterodimer or homodimer structure with an oppositely charged oridentical alpha-helix forming peptide moiety, as represented by aso-called leucine zipper peptide. The modified pilin peptide can formdimeric structures which have higher binding affinity to host cells thanthe corresponding monomer, by virtue of divalent binding, but which areless inflammatory than intact pili, due to the reduced degree ofmobilization of host-cell receptor sites, relative to that produced bybinding of intact pili. Further, the dimeric construction allows pilinpeptides from two different Pseudomonas strains to be assembled indimeric form, or a combination of a single pilin peptide and anothertherapeutic agent, e.g., an antibacterial agent carried in cleavableform on a carrier peptide which forms the other monomer in the dimericstructure.

[0055] The modified pilin peptide may be further modified, in accordancewith the invention, to reduce or eliminate immunogenicity in the targetorganism, e.g., humans.

[0056] In accordance with another aspect of the invention, thecomposition is used in treating or preventing P. aeruginosa infection ina subject a pharmaceutically effective amount of the modified P.aeruginosa pilin protein to the subject. The peptide is administered,for example, by formulating the peptide as a liquid or particulateaerosol, and delivering the aerosol to the subject's airway., or byintravenous administration.

[0057] These and other objects and features of the invention will becomemore fully apparent when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIGS. 1A-1E give the nucleotide and corresponding amino acidsequence of an exemplary truncated pilin protein from K122 (1A), PAK(1B), PAO (1C), P1 (1D, and KB7 (1E), where the nucleotide andpolypeptide sequences from K122, PAK, PAO, P1, and KB7 are identified bySEQ ID NOS: 1 and 2, SEQ ID NOS: 3 and 4, SEQ ID NOS: 5 and 6, SEQ IDNOS: 7 and 8, and SEQ ID NOS: 9 and 10, respectively;

[0059]FIGS. 2A and 2B show vector constructs designated pDLB42 andpDLB43 containing an N-terminal H coil-truncated PAK fusion peptide(2A), and an N-terminal E coil-truncated PAK fusion peptide (2B);

[0060]FIGS. 3A and 3B show the nucleotide and polypeptide sequences ofan exemplary N-terminal H coil-truncated PAK fusion peptide (3A), and anexemplary N-terminal E coil-truncated PAK fusion peptide (3B) in thevector constructs in FIGS. 2A and 2B, respectively, where the nucleotideand polypeptides sequences are identified by SEQ ID NOS: 11 and 12, andSEQ ID NOS: 13 and 14, respectively;

[0061]FIGS. 4A and 4B show nucleotide and polypeptide sequences of anexemplary N-terminal H coil-truncated K122 fusion peptide (4A), and anexemplary N-terminal E coil-truncated K122 fusion peptide, respectively,where the nucleotide and polypeptides sequences are identified by SEQ IDNOS: 15 and 16, and SEQ ID NOS: 17 and 18, respectively;

[0062]FIGS. 5A and 5B show nucleotide and polypeptide sequences of anexemplary N-terminal H coil-truncated PAO peptide (5A), and an exemplaryN-terminal E coil-truncated fusion PAO peptide, respectively, where thenucleotide and polypeptides sequences are identified by SEQ ID NOS: 19and 20, and SEQ ID NOS: 21 and 22, respectively;

[0063]FIG. 6 illustrates steps in the purification of the K122 truncatedpilin protein from FIG. 1A;

[0064]FIG. 7 is a plot of size exclusion chromatography of the truncatedK122 pilin protein from FIG. 1A;

[0065]FIG. 8 is a plot of the competitive inhibition of biotinylated PAKpili binding to immobilized asialo-GM₁ by the truncated K122 pilinprotein of FIG. 1A; and

[0066]FIG. 9 illustrates the ability of a modified pilin protein formedin accordance with the invention to prolong survival in a mouse model ofPseudomonas infection;

DETAILED DESCRIPTION OF THE INVENTION

[0067] The first section describes exemplary modified Pseudomonas pilinpeptides formed in accordance with the invention, and expression vectorsfor recombinant expression of the proteins. The second section providesprocedures for constructing a model expression vector for a truncatedPAK pilin protein, for isolating the protein, and a competitive bindingassay for characterizing the modified protein's ability to bind toASIALO-GM₁ glycosphingolipid. The method of treating or preventingPseudomonas infection, in accordance with another aspect of theinvention, and the effect of the treatment in a model animal system, isgiven in the third section.

[0068] I. Modified Pilin-Peptide Compositions

[0069] The invention takes advantage of the observation herein that theP. aeruginosa pilin protein can be modified to prevent self-assembly,i.e., oligomerization, by modifying the N terminus of the protein toprevent alpha helix formation, and the further observation herein that amonomeric or dimeric form of the pilin peptide is effective in treatingor as a prophylactic for Pseudomonas infection, but without, or with asignificantly reduced inflammatory response relative to intact pili. TheN-terminal peptide modifications contemplated herein are of threegeneral types:

[0070] (i) amino acid changes which alter the peptide's ability to formα-helical structures in the N-terminal portion of the protein,preferably in the first 2040 residues of the e.g., and (ii) deletions inthe N-terminal portion of the peptide, e.g., a deletion of the first 15to the first 40 N-terminal amino acids, preferably the first 25 to first30 N-terminal amino acids; and

[0071] (iii) replacement of the N-terminal portion with an alpha-helicalcoiled-coil homodimer or heterodimer sequence.

[0072] Amino acid modifications, e.g., substitutions, deletions, oradditions in the N-terminal portion of the peptide effective to producea non-self-assembling peptide can be determined from known physicalinteractions that determine the properties of proteins, and from theconformational properties of polypeptide chains. In particular,modifications that affect the ability of the N-terminal portion todisrupt α-helix formation in the first N-terminal 30 amino acid regionof the protein will generally be pertinent. Introduction of Proresidues, in particular, in this segment of the protein willsignificantly disrupt a-helix formation, but other residues that tend todestabilize a-helices, e.g., groups of Gly, His or Asn, are alsocontemplated (see, for example, the discussion in Proteins, supra,pages, 182-186, and refs. 35 and 36). For example, a string ofcontinuous Gly, His, or Asn residues, e.g., 3-5 residue string, willeffectively prevent alpha helix formation, as will periodic Proresidues, e.g., every 5-7 residues.

[0073] The amino acid sequences of several Pseudomonas pilin peptideshave been reported [e.g., 37-41]. Further, there is a large body ofliterature references that provide guidance as to the types andfrequency of residues that will effectively prevent alpha-helixformation are available. From these references, one may constructspecific amino acid substitutions, deletions, or additions that would bepredicted to eliminate or reduce the tendency of alpha-helix formationin the first 1540 residues of a selected pilin peptide. Alternatively, avariety of computer algorithms designed to predict secondary structuremay be employed to determine whether given amino acid substitutions inthe N-terminal region of a selected Pseudomonas pilin peptide are likelyto be effective in blocking alpha-helix formation [e.g., 25-32].

[0074] Alternatively, the N-terminal portion of the peptide may bedeleted, to produce a pilin protein whose N-terminal region is lacking acritical i-helical forming portion. As noted above, the deletions in theN-terminal portion of the peptide, are preferably the first 15 to thefirst 40 N-terminal amino acids, preferably the first 25 to first 30N-terminal amino acids. In one exemplary peptide described below, thepilin protein from strain K122 has N-terminal residues 1-28 deleted.This peptide is identified below as K-1224. The polynucleotide andcorresponding polypeptide sequences of the modified protein are given inFIG. 1A, and are identified herein as SEQ ID NO: 1 (polynucleotidesequence) and SEQ ID NO: 2 (polypeptide sequence). The first five aminoacid residues in the polypeptide sequence are not native to the K122sequence, but are derived from an intrinsic coding sequence of theexpression vector. The C-terminal residue of the polypeptide is the Proresidue immediately upstream of the two stop OCH codons; that is, thepolypeptide sequence identified by SEQ ID NO:2 does not include theresidues Ser-Ser-Lys-Leu-Gly downstream of the stop codons.

[0075] Similar polynucleotide and polypeptide sequence for truncatedpilin peptides from PAK, PAO, P1, and KB7 Pseudomonas strains are givenin FIGS. 1B-1E, respectively. The polynucleotide and polypeptidesequences for the truncated pilin peptide from strain PAK are identifiedas SEQ ID NOS:3 and 4, respectively (FIG. 1B; for the truncated pilinpeptide from strain PAO, SEQ ID NOS:5 and 6, respectively (FIG. 1C); forthe truncated pilin peptide from strain P1, SEQ ID NOS:7 and 8,respectively (FIG. 1D); and for the truncated pilin peptide from strainKB7, SEQ ID NOS:9 and 10, respectively (FIG. 1E).

[0076] The example below illustrates the recombinant production of theabove truncated pilin protein, designated K1224, truncated to delete itsN-terminal 28 amino acid residues. It will be recognized by one skilledin the art that a variety of procedures are available for producing P.aeruginosa pilin protein with a modified N-terminal region. For example,references 20-26 disclose various P. aeruginosa pilin protein genes.Reference 27 details methods for expressing pilin peptide in E. coli.These references are incorporated herein by reference.

[0077] It will be further appreciated that methods for modifying theN-terminal region of a pili protein gene, to achieve a desiredmodification in the protein, are well within the skill of personsskilled in the art. For example, site directed mutagenesis, includingsubstitution, deletion, and addition mutations of the gene sequence canbe carried out by well known methods, e.g., involving PCR primers.Similarly genes with various-length truncations can be prepared bystandard means, as exemplified below.

[0078] FIGS. 1A-1E illustrate exemplary coding sequences for N-terminalregion truncated pilin peptide. For compositions in which one or aminoacids are substituted in the first 2040 residues, to prevent alpha-helixformation in the N-terminal region, the peptide has known pilin-peptidesequences (see references above relating to P. aeruginosa pilinsequence, e.g., references 5, 12, 13, and 14), but modified to containamino acid substitutions or additions, e.g., Pro residues or Gly stringsat suitable residue positions. For producing such modified proteinsrecombinantly, one can suitably modify the coding sequence for thecorresponding pilin peptide, using standard techniques, such assite-directed mutagenesis, PCR amplification with suitable-sequenceprimers, or solid-phase synthesis.

[0079] In the third general embodiment of the composition of theinvention, the N-terminal region of a pilin peptide, e.g., the first1540 residues, is replaced by a peptide segment capable of forming acoiled-coil homodimer with an identical peptide segment, or aheterodimer with an oppositely charged peptide segment. Peptides withthis coiled-coil dimer forming property have been disclosed, e.g., inPCT applications WO 97/12988 and WO 95/31480, which are incorporatedherein by reference.

[0080] Exemplary coiled-coil peptides are referred to herein E coils,referring to negatively charged subunits whose charge is providedpredominantly by glutamic acid residues, and K coils, referring topositively charged subunits whose charge is provided dominantly bylysine residues. The two coils, when mixed, form a stable 1:1 K:E dimer.One exemplary E coil sequence is given in FIGS. 3B-5B, where the E coilsegment constitutes roughly residue numbers 13-53 of the given fusionpeptide sequences. The sequence of a K-coil sequence suitable fordimerizing with this E coil is given in the two PCT applications above.

[0081] Alternatively, the coiled-coil segment may be a homodimersequence, referred to herein as an H coil, capable of dimerizing withitself. Exemplary H coil sequences are given in FIGS. 3A-5A, where the Hcoil segment constitutes roughly residue numbers 13-53 of the givenfusion-peptide sequences. When mixed, these two segment form a 1:1 H:Hhomodimer.

[0082] To produce a heterodimer modified pilin peptide, fusion proteinscontaining both E-coil and K-coil N-terminal segments are formed, thenmixed to produce the desired dimer. The two different peptides formingthe dimer may be modified pilin protein from the same strain, e.g., aPAK/PAK pilin dimer, or from two different strains, e.g., a PAK/K122dimer. Alternatively, one of the two peptides may be a non-pilin relatedpeptide, for example, a carrier protein that is itself a therapeuticpeptide, e.g., peptide anti-bacterial agent, or a carrier proteinderivatized with a therapeutic compound that can be cleaved from thecarrier, e.g., by an esterase.

[0083] In the case of a homodimer, the two modified pilin proteins willin general be same, although homodimers with different strain pilinproteins or with a mixture of a pilin and non-pilin peptide, can beformed in a mixture of same-peptide and different-peptide dimers.

[0084]FIGS. 2A and 2B show the general construction of expressionvectors for recombinant production in an E. coli host of modified pilinpeptides (fusion peptides) with an N-terminal H coil-truncated pilinfusion peptide, in this case, the PAK peptide (2A), and an N-terminal Ecoil-truncated PAK pilin fusion peptide 2B). The vectors are constructedaccording to standard procedures, by inserting a suitable codingsequence into the pRLD vector cut with EcoR1 and HindIII [33, 34]. Thepolynucleotide sequence given in FIGS. 3-5 illustrate exemplary codingsequences for the fusion proteins that are inserted into the vectors.

[0085]FIGS. 3A and 3B show the nucleotide and polypeptide sequences ofthe N-terminal H coil-truncated PAK peptide (3A), and the N-terminal Ecoil-truncated PAK peptide (3B) in the vector constructs in FIGS. 2A and2B, respectively. The nucleotide and polypeptides sequences areidentified by SEQ ID NOS: 11 and 12 (H coil peptide), and SEQ ID NOS: 13and 14 (E coil peptide), respectively. FIGS. 4A and 4B show nucleotideand polypeptide sequences for the H-coil and E-coil fusion proteins,respectively, where the sequences are identified by SEQ ID NOS: 15 and16 (H-coil peptide), and SEQ ID NOS: 17 and 18 (E coil peptide),respectively. FIGS. 5A and 5B show nucleotide and polypeptide sequencesof the N-terminal H coil-truncated PAO peptide (5A), and the N-terminalE coil-truncated PAO peptide (5B), respectively, where the nucleotideand polypeptides sequences are identified by SEQ ID NOS: 19 and 20 (Hcoil peptide), and SEQ ID NOS: 21 and 22 (E coil peptide, respectively).

[0086] The modified pilin peptide of the invention may be furthermodified to reduce or eliminate its immunogenicity in humans. This canbe done, for example, following the approach disclosed in PCTapplication WO 98/52976, which is incorporated herein by reference.Briefly, the approach involves the steps of (a) determining at leastpart of the amino acid sequence of the protein, in this case, modifiedpilin peptide, (b) identifying in the amino acid sequence one or morepotential epitopes for T-cells (T-cell epitopes) of the given species;and (c) modifying the amino acid sequence to eliminate at least one ofthe T-cell epitopes identified in step (b) thereby to eliminate orreduce the immunogenicity of the protein when exposed to the immunesystem of the given species.

[0087] II. Production and Characterization of Modified Pilin Proteins

[0088] A. Preparing the Coding Sequence and Construction of ExpressionVector.

[0089] In one general method, modified pilin proteins are prepared byPCR amplification of known and available pilin coding sequences usingprimers that effect the desired deletion, modification or insertion of acoiled-coil moiety in the amplified coding sequences. The primers alsoprovide suitable endonuclease cutting sequences at the amplifiedfragment termini for introduction into selected insertion sites of anexpression vector. After amplification and endonuclease treatment, thecoding sequence fragment is purified and placed in a suitable expressionvector, e.g., an E coli expression vector, under the control od asuitable promoter, for host-cell expression. Example 1 below detailsconstruction of the coding sequence and an expression vector for thetruncated PAK pilin peptide whose sequence in shown in FIG. 1A.

EXAMPLE 1

[0090] Polymerase Chain Reaction (PCR).

[0091] PCR was performed in Stratagene Robocycler 40, thermocycler,using the standard protocol. Each reaction mixture (a total of 10 ul)containing the reaction buffer 700 mM Tris HCL pH8.8, 200 mM MgCl₂, 200uM each dNTP, template DNA (1 ug), 825 ng of each of the primers and 2.5units of Taq polymerase were denatured for 10 min at 94° C., followed,by 30 amplification cycles (3 min denaturation, at 94° C., 2 minannealing at 58° C. and 2 min extension at 72° C.

[0092] DNA sequencing.

[0093] DNA from the cloned plasmid preparations were sequenced using thedideoxy nucleotide method of Sanger et al., in combination withappropriate oligonucleotides used as primers.

[0094] Truncated K1224 Pilin Protein Gene.

[0095] Truncated K122 (1-28) pilin gene was engineered by usingpolymerase chain reaction (PCR). First, previously cloned K122 DNA (22)containing pilin gene was subjected to PCR using the syntheticoligonucleotides primers (with restriction sites added for cloningpurposes) flanking the beginning and end of the nucleotide residuescorresponding to the amino acids 28 and 150. The resulting PCR productwas purified by electrophoresis on an 8% polyacrylamide gel. Fragmentsof about 380 bp were isolated, and digested with Ecor1 and HindIIIenzymes. The digests were purified by Phenol-extraction followed byethanol precipitation. FIG. 1A shows the polynucleotide sequence, andcorresponding amino acid sequence of the truncated pilin protein.

[0096] The purified digests were cloned into pRLD expression vector atEcor1-HindiIII sites. The ligated plasmid DNA was transformed into anexpression host BL21 strain. Plasmid DNAs were isolated from theCarbenicillin resistant recombinants by the cleared lysate method, anddigested with restriction enzymes to check for the correct size inserts.Recombinant DNA containing the correct size inserts were sequenced fromboth the orientations as described above.

[0097] B. Expression of Modified Pilin Protein.

[0098] The recombinant protein is expressed in a suitable host undersuitable expression conditions, according to well-known methods. Forexample, for bacterial synthesis, the protein may be obtained in theperiplasmic space of the bacteria, or in secreted form in the host-cellculture medium. Example 2 below illustrates the expression of the abovetruncated PAK pilin protein in E. coli.

EXAMPLE 2

[0099]E. coli cells (BL21) harboring the K122(1-28) plasmid containingK122 truncated pilin gene were grown at 37° C., with shaking in LBmedium containing carbenicillin (100 μg/ml) to an A50 of 0.5-0.7.Production of recombinant protein was induced by the addition ofisopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1mM. The bacteria were then grown for an additional 8 hours at 37° C. Theexpressed periplasmic protein was extracted by osmotic shock as follows:The cells were harvested at 4,000g for 10 min, at 4° C. and re-suspendedin TES buffer (10 mM Tris-HCl, 5 mM EDTA, 20% sucrose, pH 8.0.) in afinal volume of 80 ml per gram of wet weight. Cells were shaken gentlyat room temp (150 rpm) for 10 min. The suspension was then centrifugedand the resulting pellet was re-suspended in 5 mM ice-cold MgSO₄ (80 mlper gram of wet weight). The cell suspension was shaken gently for 30min on ice, and subsequently centrifuged at 8,000 g for 15 min at 4° C.The supernatant continuing the periplasmic fraction was then furtherclarified by passing through a 0.45 μm filter and subsequently purifiedby the column chromatography as described.

[0100] C. Protein Purification.

[0101] Methods that have been reported for purification of Pseudomonaspilin peptide are suitable, although some modification to accommodatethe modified sequence may be required. In the case of a fusion pilinpeptide having an N-terminal coiled-coil peptide moiety, the fusionprotein can be isolated by affinity chromatography, using an immobilizedcoiled-coil peptide to capture the fusion protein. FIG. 6 illustrates ageneral scheme for purifying a modified pilin protein formed inaccordance with the invention, as detailed in Example 3 below. It willbe appreciated that the protein purification scheme is exemplary only.

EXAMPLE 3

[0102] The periplasmic fractions from the transformed bacterialexpression host cells were filtered through 0.45 μm filter and dilutedwith an equal volume of 20 mM sodium acetate pH 4.5 buffer and thenadsorbed to a carboxymethyl-cellulose column (CM-52 of 30 cm×2 cm) whichhas been previously equilibrated with 10 mM sodium acetate pH 4.5 buffer(base buffer) and eluted with a linear gradient of 0-0.8M NaCl in 10 mMsodium acetate pH 4.5. Fractions (3 ml volume) were collected and theabsorbance at A280 nm determined. Fractions containing pilin proteinwere pooled, freeze dried and dissolved in small amounts of distilledwater, and further fractionated on a Sephadex G-75.

[0103] As seen in FIG. 7, the molecular weight of the pilin protein(star on the plot) was about 13 KD, consistent with the 129 amino acidresidue length of the protein (see FIG. 1A).

[0104] The isolated protein was fractionated by electrophoresis onsodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGEgels). Gels were subsequently stained with Coomassie brilliant blueR-250 or transferred to PVDF membranes. Membranes were incubated withK122 IgG antiserum. The secondary antibody used was anti-mouse IgGalkaline-phosphatase. Bound antibody was detected with BCIP substrate.The results (not shown) indicate a substantially pure protein.

[0105] D. Ability of Fusion Protein to Compete with Native Pili forBinding to Receptor Sites.

[0106] To confirm that the modified pilin peptide, including dimerizedforms of the peptide, are capable of competing with pili for binding toreceptor sites, the peptide may be tested in a competitive binding assaywith native pili. The pili may be from same-strain or different-strainPseudomonas organisms. Further to this point, the modified pilin can betested against a number of different-strain pili, to test thecross-specificity the peptide is likely to have as a therapeutic agent.Example 4 below describes an exemplary binding assay of this type.

EXAMPLE 4

[0107] A polystyrene microtitre plate (Costar, Cambridge, Mass.) wascoated with 50 μl/well of asialo-GM₁ (40 μg/ml) in methanol. The solventwas evaporated at room temperature inside a fumehood. Non-specificbinding sites were blocked with 200 μl/well of 5% (w/v) BSA in PBS.After incubating at 37° C. for 1.5 hours, the wells were washed 3 timeswith 250 μl of PBS supplemented with 0.05% (w/v) BSA (Buffer A).Aliquots (50 μl) of biotinylated P. aeruginosa PAK pili (0.88 mg/ml,diluted 1:1000 in Buffer A) containing various concentrations of K1224truncated pilin were added to each well. After a 2 hour incubation at37° C. the wells were washed 5 times with 250 μl of Buffer A, then 50μl/well of streptavidin-alkaline phosphatase conjugate (Gibco BRL) at1:3000 dilution with Buffer A was added and incubated for 1 hour at roomtemperature. Following incubation, the plate was washed 5 times with 250μl/well of Buffer A. Following washing 80 μl/well ofp-nitrophenylphosphate substrate solution (1 mg/mil in 10%diethanolamine, pH 9.8) was then added. Readings at 405 nm were recordedand the results were expressed as percent inhibition. As seen in FIG.8., the percent inhibition was dose dependent on the concentration ofthe truncated protein.

[0108] III. Treatment Method

[0109] The modified pilin peptide composition of the invention is usefulin treating existing Pseudomonas infection, or as a prophylactictreatment for an individual at risk of Pseudomonas infection, e.g.,cystic fibrosis patients, burn patients, and severe neutropenic patients(e.g., cancer patients receiving chemotherapy) and intensive care unitpatients receiving respiratory support.

[0110] The peptide that is administered in the method may be modified inits N-terminal segment by deletion, substitution, or dimer-formingmoieties, as detailed above. Further, the peptide may be modified, e.g.,as detailed in WO98/52976 for reduced immunogenicity.

[0111] One preferred method of administration is by inhalation,typically in an aerosolized or microparticle form. Methods for preparingand administering peptides by aerosol are well known and suitable forthis method. Typically, the amount of peptide administered is betweenabout 0.5 to 25 mg/dose/patient, with the amount of peptide reaching thepulmonary airways depending on the efficiency of the aerosol andadministration procedure. The peptide may be administered periodically,e.g., every 68 hours, over a period until a satisfactory therapeutic endpoint is reached.

[0112] Alternatively, the peptide may be administered by transmucosalroute, e.g., intranasally, or through intravenous (IV), intraperitoneal(IP), intramuscular (IM), or subcutaneous (SubQ) injection.

[0113] Typically, doses in an amount of 1 mg to 50 mg, administered oncea day or over a more frequent dosing schedule, are suitable, althoughhigher doses may be required for IP, IM, or SubQ administration, do tothe relatively slow peptide release and uptake at sites if infection.Transdermal administration may also be effective assuming that thepeptide can be taken up efficiently by this route. Example 5 belowdemonstrates therapeutic efficacy when the peptide is administeredintraperitoneally.

[0114] For prophylactic administration, the peptide may be administeredin a single dose, or multiple doses, e.g., every 8 hours, for a periodpreceding elevated risk of infection, at peptide doses similar to thosegiven above.

[0115] In the treatment method, the peptide may be administered inconjunction with anti-bacterial agents, typically non-peptide agents,conventionally used to treat Pseudomonas infection.

EXAMPLE 5

[0116] A.BY/SnJ mice were used as they are less resistant to P.aeruginosa infection than other mouse strains. Weight: 18-20 grams; age:10 weeks. K1224 truncated pilin (FIG. 1A) was administeredintraperitoneally to A.BY/SnJ mice 15 minutes prior to the mice beingchallenged intraperitoneally with PAK wildtype at 3LD₅₀ Mice weremonitored on a hourly basis between 16 and 48 hours. As seen, percentsurvival was dose dependent within the range of pilin protein amountstested.

[0117] From the foregoing, it can be seen that (i) an N-terminalmodified pilin protein unable to oligomerize (self-assemble) is readilyexpressed as a secreted processed protein in a recombinant expressionsystem; (ii) the modified protein retains a functional epithelial cellreceptor binding domain that mediates binding to GalNAcGal containingglycoconjugates; (iii) the modified protein is monomeric at proteinconcentrations of <600 μg/ml; and (iv), pre-administration of themodified protein confers a dose-dependent protection from challenge witha heterologous P. aeruginosa strain in mammals.

[0118] Although the invention has been described with respect tospecific embodiments, it will be appreciated that the a variety ofdifferent pilin peptide N-terminal modifications, and a variety ofdifferent P. aeruginosa strains may be employed without departing fromthe invention.

Sequence Listing

[0119] SEQ ID NO: 1: polynucleotide sequence of modified K122 pilinpeptide;

[0120] SEQ ID NO: 2 polypeptide sequence of modified K122 pilin peptide;

[0121] SEQ ID NO: 3: polynucleotide sequence of modified PAK pilinpeptide;

[0122] SEQ ID NO: 4 polypeptide sequence of modified PAK pilin peptide;

[0123] SEQ ID NO: 5: polynucleotide sequence of modified PAO pilinpeptide;

[0124] SEQ ID NO: 6: polypeptide sequence of modified PAO pilin peptide;

[0125] SEQ ID NO: 7: polynucleotide sequence of modified P1 pilinpeptide;

[0126] SEQ ID NO: 8: polypeptide sequence of modified P1 pilin peptide;

[0127] SEQ ID NO: 9: polynucleotide sequence of modified KB7 pilinpeptide;

[0128] SEQ ID NO: 10: polypeptide sequence of modified KB7 pilinpeptide;

[0129] SEQ ID NO: 11: polynucleotide sequence of H-coil/truncated PAKpilin peptide;

[0130] SEQ ID NO: 12: polypeptide sequence of H-coil/truncated PAK pilinpeptide;

[0131] SEQ ID NO: 13: polynucleotide sequence of E-coil/truncated PAKpilin peptide;

[0132] SEQ ID NO: 14: polypeptide sequence of E-coil/truncated PAK pilinpeptide;

[0133] SEQ ID NO: 15: polynucleotide sequence of H-coil/truncated K122pilin peptide;

[0134] SEQ ID NO: 16: polypeptide sequence of H-coil/truncated K122pilin peptide;

[0135] SEQ ID NO: 17: polynucleotide sequence of E-coil/truncated K122pilin peptide;

[0136] SEQ ID NO: 18: polypeptide sequence of E-coil/truncated K122pilin peptide;

[0137] SEQ ID NO: 19: polynucleotide sequence of H-coil/truncated PAOpilin peptide;

[0138] SEQ ID NO: 20: polypeptide sequence of H-coil/truncated PAO pilinpeptide;

[0139] SEQ ID NO: 21: polynucleotide sequence of E-coil/truncated PAOpilin peptide;

[0140] SEQ ID NO: 22: polypeptide sequence of E-coil/truncated PAO pilinpeptide;

1 22 1 387 DNA Pseudomonas aeruginosa CDS (0)...(0) 1 gcgctcgagggtaccgaatt cgcacgcgct cagcttagcg aacgcatgac cctggccagt 60 ggtctcaagacgaaagtgag cgatatcttc tctcaggatg ggtcctgccc ggctaatact 120 gctgccacggcaggcatcga gaaagatacc gacatcaacg gcaagtatgt tgccaaggta 180 acaactggtggcaccgcagc tgcgtctggt ggttgcacta tcgttgctac tatgaaagcc 240 tctgatgtggctactcctct gagggggaaa actctgactt tgactctagg aaatgctgac 300 aagggttcttacacttgggc ctgtacttcc aacgcagata acaagtacct gccaaaaacc 360 tgccagactgctaccactac cactccg 387 2 129 PRT Pseudomonas aeruginosa 2 Ala Leu GluGly Thr Glu Phe Ala Arg Ala Gln Leu Ser Glu Arg Met 1 5 10 15 Thr LeuAla Ser Gly Leu Lys Thr Lys Val Ser Asp Ile Phe Ser Gln 20 25 30 Asp GlySer Cys Pro Ala Asn Thr Ala Ala Thr Ala Gly Ile Glu Lys 35 40 45 Asp ThrAsp Ile Asn Gly Lys Tyr Val Ala Lys Val Thr Thr Gly Gly 50 55 60 Thr AlaAla Ala Ser Gly Gly Cys Thr Ile Val Ala Thr Met Lys Ala 65 70 75 80 SerAsp Val Ala Thr Pro Leu Arg Gly Lys Thr Leu Thr Leu Thr Leu 85 90 95 GlyAsn Ala Asp Lys Gly Ser Tyr Thr Trp Ala Cys Thr Ser Asn Ala 100 105 110Asp Asn Lys Tyr Leu Pro Lys Thr Cys Gln Thr Ala Thr Thr Thr Thr 115 120125 Pro 3 369 DNA Pseudomonas aeruginosa 3 gcgctcgagg gtaccgaattcgctcgttcg gaaggcgcat ctgctcttgc ttcggtcaat 60 ccgttgaaga ctaccgttgaagaggcgctt tctcgtggtt ggagcgtgaa gagcggtaca 120 ggtacagagg acgctactaagaaagaggtt cctctggggg tggcggcaga tgctaacaaa 180 ctgggtacta tcgcactcaaacccgatcct gctgatggta ctgcagatat cactttgact 240 ttcactatgg gcggtgcaggaccgaagaat aaagggaaaa ttattaccct gactcgtact 300 gcagctgatg gtctctggaagtgcaccagt gatcaggatg agcagtttat tccgaaaggt 360 tgctctagg 369 4 123 PRTPseudomonas aeruginosa 4 Ala Leu Glu Gly Thr Glu Phe Ala Arg Ser Glu GlyAla Ser Ala Leu 1 5 10 15 Ala Ser Val Asn Pro Leu Lys Thr Thr Val GluGlu Ala Leu Ser Arg 20 25 30 Gly Trp Ser Val Lys Ser Gly Thr Gly Thr GluAsp Ala Thr Lys Lys 35 40 45 Glu Val Pro Leu Gly Val Ala Ala Asp Ala AsnLys Leu Gly Thr Ile 50 55 60 Ala Leu Lys Pro Asp Pro Ala Asp Gly Thr AlaAsp Ile Thr Leu Thr 65 70 75 80 Phe Thr Met Gly Gly Ala Gly Pro Lys AsnLys Gly Lys Ile Ile Thr 85 90 95 Leu Thr Arg Thr Ala Ala Asp Gly Leu TrpLys Cys Thr Ser Asp Gln 100 105 110 Asp Glu Gln Phe Ile Pro Lys Gly CysSer Arg 115 120 5 366 DNA Pseudomonas aeruginosa CDS (0)...(0) 5gcgctcgagg gtaccgaatt cgcgcgttcg gaaggtgctt cggcgctggc gacgatcaac 60ccgctgaaga ccactgttga agagtcgctg tcgcgtggaa ttgctggtag caaaattaaa 120attggtacta ctgcttctac tgcgaccgaa acatatgccg gcgtcgagcc ggatgccaac 180aagttgggtg taattgctgt agcaatcgaa gatagtggtg cgggtgatat tacctttacc 240ttccagactg gtacctctag tcccaagaat gctactaaag ttatcactct gaaccgtact 300gcggatgggg tctgggcttg taaatctacc caggatccga tgttcactcc gaaaggttct 360gataac 366 6 122 PRT Pseudomonas aeruginosa 6 Ala Leu Glu Gly Thr GluPhe Ala Arg Ser Glu Gly Ala Ser Ala Leu 1 5 10 15 Ala Thr Ile Asn ProLeu Lys Thr Thr Val Glu Glu Ser Leu Ser Arg 20 25 30 Gly Ile Ala Gly SerLys Ile Lys Ile Gly Thr Thr Ala Ser Thr Ala 35 40 45 Thr Glu Thr Tyr AlaGly Val Glu Pro Asp Ala Asn Lys Leu Gly Val 50 55 60 Ile Ala Val Ala IleGlu Asp Ser Gly Ala Gly Asp Ile Thr Phe Thr 65 70 75 80 Phe Gln Thr GlyThr Ser Ser Pro Lys Asn Ala Thr Lys Val Ile Thr 85 90 95 Leu Asn Arg ThrAla Asp Gly Val Trp Ala Cys Lys Ser Thr Gln Asp 100 105 110 Pro Met PheThr Pro Lys Gly Ser Asp Asn 115 120 7 381 DNA Pseudomonas aeruginosa CDS(0)...(0) 7 gcgctcgagg gtaccgaatt cgcccgtacc caggtgaccc gtgccgtgagtgaagtcagc 60 gcgctgaaga ccgctgcgga gtcggcgatt ctggaaggga aggagattgtttccagcgcg 120 actcctaaag atacccagta tgacattggc ttcaccgagt ctactttgctagatggttct 180 ggtaagagtc agatccaggt aacggacaat aaagatggca ccgttgagttggtcgctacc 240 ttgggtaaat cttctggttc cgccatcaaa ggggctgtaa tcactgtttcgcgtaaaaat 300 gacggagtct ggaactgcaa aatcaccaaa actcctacag cttggaagcccaactacgct 360 ccggctaatt gcccgaattc c 381 8 127 PRT Pseudomonasaeruginosa 8 Ala Leu Glu Gly Thr Glu Phe Ala Arg Thr Gln Val Thr Arg AlaVal 1 5 10 15 Ser Glu Val Ser Ala Leu Lys Thr Ala Ala Glu Ser Ala IleLeu Glu 20 25 30 Gly Lys Glu Ile Val Ser Ser Ala Thr Pro Lys Asp Thr GlnTyr Asp 35 40 45 Ile Gly Phe Thr Glu Ser Thr Leu Leu Asp Gly Ser Gly LysSer Gln 50 55 60 Ile Gln Val Thr Asp Asn Lys Asp Gly Thr Val Glu Leu ValAla Thr 65 70 75 80 Leu Gly Lys Ser Ser Gly Ser Ala Ile Lys Gly Ala ValIle Thr Val 85 90 95 Ser Arg Lys Asn Asp Gly Val Trp Asn Cys Lys Ile ThrLys Thr Pro 100 105 110 Thr Ala Trp Lys Pro Asn Tyr Ala Pro Ala Asn CysPro Asn Ser 115 120 125 9 381 DNA Pseudomonas aeruginosa CDS (0)...(0) 9gcgctcgagg gtaccgaatt ctctcgctct caggtctcca gggttatggc ggaggctggc 60tccttgaaga ctgcagttga ggcctgcctc caggatggtc gtactgctgt gggtactgct 120gctggtcaat gcgatccggg tgcgacgggt tccagtttgt tgactggtgc ttctcagact 180tctcaaaccc tgccaaccaa taccggtgtt ccgcaggttc tggatcctct gactactcaa 240accactatca ttgcgacttt tggtaacggc gcatccgcag ctatttctgg ccagactctg 300acctggactc gtgatgttaa tggtggctgg agctgtgcta ctaccgtaga tgctaaattc 360cgtcctaatg gctgtactga c 381 10 127 PRT Pseudomonas aeruginosa 10 Ala LeuGlu Gly Thr Glu Phe Ser Arg Ser Gln Val Ser Arg Val Met 1 5 10 15 AlaGlu Ala Gly Ser Leu Lys Thr Ala Val Glu Ala Cys Leu Gln Asp 20 25 30 GlyArg Thr Ala Val Gly Thr Ala Ala Gly Gln Cys Asp Pro Gly Ala 35 40 45 ThrGly Ser Ser Leu Leu Thr Gly Ala Ser Gln Thr Ser Gln Thr Leu 50 55 60 ProThr Asn Thr Gly Val Pro Gln Val Leu Asp Pro Leu Thr Thr Gln 65 70 75 80Thr Thr Ile Ile Ala Thr Phe Gly Asn Gly Ala Ser Ala Ala Ile Ser 85 90 95Gly Gln Thr Leu Thr Trp Thr Arg Asp Val Asn Gly Gly Trp Ser Cys 100 105110 Ala Thr Thr Val Asp Ala Lys Phe Arg Pro Asn Gly Cys Thr Asp 115 120125 11 507 DNA Pseudomonas aeruginosa CDS (0)...(0) 11 gcgctcgagcaccatcatca ccatggtggt ggtggcgaga ttgaggccct caaggctgaa 60 atcgaagccctaaaggccga gatagaagca cttaaggcag agatcgaggc gctaaaagcg 120 gaaatagaggctctgaaggc aggcggtgga ggagaattcg ctcgttcgga aggcgcatct 180 gctcttgcttcggtcaatcc gttgaagact accgttgaag aggcgctttc tcgtggttgg 240 agcgtgaagagcggtacagg tacagaggac gctactaaga aagaggttcc tctgggggtg 300 gcggcagatgctaacaaact gggtactatc gcactcaaac ccgatcctgc tgatggtact 360 gcagatatcactttgacttt cactatgggc ggtgcaggac cgaagaataa agggaaaatt 420 attaccctgactcgtactgc agctgatggt ctctggaagt gcaccagtga tcaggatgag 480 cagtttattccgaaaggttg ctctagg 507 12 169 PRT Pseudomonas aeruginosa 12 Ala Leu GluHis His His His His Gly Gly Gly Gly Glu Ile Glu Ala 1 5 10 15 Leu LysAla Glu Ile Glu Ala Leu Lys Ala Glu Ile Glu Ala Leu Lys 20 25 30 Ala GluIle Glu Ala Leu Lys Ala Glu Ile Glu Ala Leu Lys Ala Gly 35 40 45 Gly GlyGly Glu Phe Ala Arg Ser Glu Gly Ala Ser Ala Leu Ala Ser 50 55 60 Val AsnPro Leu Lys Thr Thr Val Glu Glu Ala Leu Ser Arg Gly Trp 65 70 75 80 SerVal Lys Ser Gly Thr Gly Thr Glu Asp Ala Thr Lys Lys Glu Val 85 90 95 ProLeu Gly Val Ala Ala Asp Ala Asn Lys Leu Gly Thr Ile Ala Leu 100 105 110Lys Pro Asp Pro Ala Asp Gly Thr Ala Asp Ile Thr Leu Thr Phe Thr 115 120125 Met Gly Gly Ala Gly Pro Lys Asn Lys Gly Lys Ile Ile Thr Leu Thr 130135 140 Arg Thr Ala Ala Asp Gly Leu Trp Lys Cys Thr Ser Asp Gln Asp Glu145 150 155 160 Gln Phe Ile Pro Lys Gly Cys Ser Arg 165 13 507 DNAPseudomonas aeruginosa CDS (0)...(0) 13 gcgctcgagc accatcatca ccatggtggtggtggcgagg tatccgcttt agagaaagaa 60 gtttctgctc tcgaaaaaga ggtcagtgctctggaaaaag aggtgtcagc cttggaaaag 120 gaagtatcag cacttgagaa gggcggtggaggagaattcg ctcgttcgga aggcgcatct 180 gctcttgctt cggtcaatcc gttgaagactaccgttgaag aggcgctttc tcgtggttgg 240 agcgtgaaga gcggtacagg tacagaggacgctactaaga aagaggttcc tctgggggtg 300 gcggcagatg ctaacaaact gggtactatcgcactcaaac ccgatcctgc tgatggtact 360 gcagatatca ctttgacttt cactatgggcggtgcaggac cgaagaataa agggaaaatt 420 attaccctga ctcgtactgc agctgatggtctctggaagt gcaccagtga tcaggatgag 480 cagtttattc cgaaaggttg ctctagg 50714 169 PRT Pseudomonas aeruginosa 14 Ala Leu Glu His His His His His GlyGly Gly Gly Glu Val Ser Ala 1 5 10 15 Leu Glu Lys Glu Val Ser Ala LeuGlu Lys Glu Val Ser Ala Leu Glu 20 25 30 Lys Glu Val Ser Ala Leu Glu LysGlu Val Ser Ala Leu Glu Lys Gly 35 40 45 Gly Gly Gly Glu Phe Ala Arg SerGlu Gly Ala Ser Ala Leu Ala Ser 50 55 60 Val Asn Pro Leu Lys Thr Thr ValGlu Glu Ala Leu Ser Arg Gly Trp 65 70 75 80 Ser Val Lys Ser Gly Thr GlyThr Glu Asp Ala Thr Lys Lys Glu Val 85 90 95 Pro Leu Gly Val Ala Ala AspAla Asn Lys Leu Gly Thr Ile Ala Leu 100 105 110 Lys Pro Asp Pro Ala AspGly Thr Ala Asp Ile Thr Leu Thr Phe Thr 115 120 125 Met Gly Gly Ala GlyPro Lys Asn Lys Gly Lys Ile Ile Thr Leu Thr 130 135 140 Arg Thr Ala AlaAsp Gly Leu Trp Lys Cys Thr Ser Asp Gln Asp Glu 145 150 155 160 Gln PheIle Pro Lys Gly Cys Ser Arg 165 15 525 DNA Pseudomonas aeruginosa CDS(0)...(0) 15 gcgctcgagc accatcatca ccatggtggt ggtggcgaga ttgaggccctcaaggctgaa 60 atcgaagccc taaaggccga gatagaagca cttaaggcag agatcgaggcgctaaaagcg 120 gaaatagagg ctctgaaggc aggcggtgga ggagaattcg cacgcgctcagcttagcgaa 180 cgcatgaccc tggccagtgg tctcaagacg aaagtgagcg atatcttctctcaggatggg 240 tcctgcccgg ctaatactgc tgccacggca ggcatcgaga aagataccgacatcaacggc 300 aagtatgttg ccaaggtaac aactggtggc accgcagctg cgtctggtggttgcactatc 360 gttgctacta tgaaagcctc tgatgtggct actcctctga gggggaaaactctgactttg 420 actctaggaa atgctgacaa gggttcttac acttgggcct gtacttccaacgcagataac 480 aagtacctgc caaaaacctg ccagactgct accactacca ctccg 525 16175 PRT Pseudomonas aeruginosa 16 Ala Leu Glu His His His His His GlyGly Gly Gly Glu Ile Glu Ala 1 5 10 15 Leu Lys Ala Glu Ile Glu Ala LeuLys Ala Glu Ile Glu Ala Leu Lys 20 25 30 Ala Glu Ile Glu Ala Leu Lys AlaGlu Ile Glu Ala Leu Lys Ala Gly 35 40 45 Gly Gly Gly Glu Phe Ala Arg AlaGln Leu Ser Glu Arg Met Thr Leu 50 55 60 Ala Ser Gly Leu Lys Thr Lys ValSer Asp Ile Phe Ser Gln Asp Gly 65 70 75 80 Ser Cys Pro Ala Asn Thr AlaAla Thr Ala Gly Ile Glu Lys Asp Thr 85 90 95 Asp Ile Asn Gly Lys Tyr ValAla Lys Val Thr Thr Gly Gly Thr Ala 100 105 110 Ala Ala Ser Gly Gly CysThr Ile Val Ala Thr Met Lys Ala Ser Asp 115 120 125 Val Ala Thr Pro LeuArg Gly Lys Thr Leu Thr Leu Thr Leu Gly Asn 130 135 140 Ala Asp Lys GlySer Tyr Thr Trp Ala Cys Thr Ser Asn Ala Asp Asn 145 150 155 160 Lys TyrLeu Pro Lys Thr Cys Gln Thr Ala Thr Thr Thr Thr Pro 165 170 175 17 525DNA Pseudomonas aeruginosa CDS (0)...(0) 17 gcgctcgagc accatcatcaccatggtggt ggtggcgagg tatccgcttt agagaaagaa 60 gtttctgctc tcgaaaaagaggtcagtgct ctggaaaaag aggtgtcagc cttggaaaag 120 gaagtatcag cacttgagaagggcggtgga ggagaattcg cacgcgctca gcttagcgaa 180 cgcatgaccc tggccagtggtctcaagacg aaagtgagcg atatcttctc tcaggatggg 240 tcctgcccgg ctaatactgctgccacggca ggcatcgaga aagataccga catcaacggc 300 aagtatgttg ccaaggtaacaactggtggc accgcagctg cgtctggtgg ttgcactatc 360 gttgctacta tgaaagcctctgatgtggct actcctctga gggggaaaac tctgactttg 420 actctaggaa atgctgacaagggttcttac acttgggcct gtacttccaa cgcagataac 480 aagtacctgc caaaaacctgccagactgct accactacca ctccg 525 18 175 PRT Pseudomonas aeruginosa 18 AlaLeu Glu His His His His His Gly Gly Gly Gly Glu Val Ser Ala 1 5 10 15Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu 20 25 30Lys Glu Val Ser Ala Leu Glu Lys Glu Val Ser Ala Leu Glu Lys Gly 35 40 45Gly Gly Gly Glu Phe Ala Arg Ala Gln Leu Ser Glu Arg Met Thr Leu 50 55 60Ala Ser Gly Leu Lys Thr Lys Val Ser Asp Ile Phe Ser Gln Asp Gly 65 70 7580 Ser Cys Pro Ala Asn Thr Ala Ala Thr Ala Gly Ile Glu Lys Asp Thr 85 9095 Asp Ile Asn Gly Lys Tyr Val Ala Lys Val Thr Thr Gly Gly Thr Ala 100105 110 Ala Ala Ser Gly Gly Cys Thr Ile Val Ala Thr Met Lys Ala Ser Asp115 120 125 Val Ala Thr Pro Leu Arg Gly Lys Thr Leu Thr Leu Thr Leu GlyAsn 130 135 140 Ala Asp Lys Gly Ser Tyr Thr Trp Ala Cys Thr Ser Asn AlaAsp Asn 145 150 155 160 Lys Tyr Leu Pro Lys Thr Cys Gln Thr Ala Thr ThrThr Thr Pro 165 170 175 19 504 DNA Pseudomonas aeruginosa CDS (0)...(0)19 gcgctcgagc accatcatca ccatggtggt ggtggcgaga ttgaggccct caaggctgaa 60atcgaagccc taaaggccga gatagaagca cttaaggcag agatcgaggc gctaaaagcg 120gaaatagagg ctctgaaggc aggcggtgga ggagaattcg cgcgttcgga aggtgcttcg 180gcgctggcga cgatcaaccc gctgaagacc actgttgaag agtcgctgtc gcgtggaatt 240gctggtagca aaattaaaat tggtactact gcttctactg cgaccgaaac atatgccggc 300gtcgagccgg atgccaacaa gttgggtgta attgctgtag caatcgaaga tagtggtgcg 360ggtgatatta cctttacctt ccagactggt acctctagtc ccaagaatgc tactaaagtt 420atcactctga accgtactgc ggatggggtc tgggcttgta aatctaccca ggatccgatg 480ttcactccga aaggttctga taac 504 20 168 PRT Pseudomonas aeruginosa 20 AlaLeu Glu His His His His His Gly Gly Gly Gly Glu Ile Glu Ala 1 5 10 15Leu Lys Ala Glu Ile Glu Ala Leu Lys Ala Glu Ile Glu Ala Leu Lys 20 25 30Ala Glu Ile Glu Ala Leu Lys Ala Glu Ile Glu Ala Leu Lys Ala Gly 35 40 45Gly Gly Gly Glu Phe Ala Arg Ser Glu Gly Ala Ser Ala Leu Ala Thr 50 55 60Ile Asn Pro Leu Lys Thr Thr Val Glu Glu Ser Leu Ser Arg Gly Ile 65 70 7580 Ala Gly Ser Lys Ile Lys Ile Gly Thr Thr Ala Ser Thr Ala Thr Glu 85 9095 Thr Tyr Ala Gly Val Glu Pro Asp Ala Asn Lys Leu Gly Val Ile Ala 100105 110 Val Ala Ile Glu Asp Ser Gly Ala Gly Asp Ile Thr Phe Thr Phe Gln115 120 125 Thr Gly Thr Ser Ser Pro Lys Asn Ala Thr Lys Val Ile Thr LeuAsn 130 135 140 Arg Thr Ala Asp Gly Val Trp Ala Cys Lys Ser Thr Gln AspPro Met 145 150 155 160 Phe Thr Pro Lys Gly Ser Asp Asn 165 21 504 DNAPseudomonas aeruginosa CDS (0)...(0) 21 gcgctcgagc accatcatca ccatggtggtggtggcgagg tatccgcttt agagaaagaa 60 gtttctgctc tcgaaaaaga ggtcagtgctctggaaaaag aggtgtcagc cttggaaaag 120 gaagtatcag cacttgagaa gggcggtggaggagaattcg cgcgttcgga aggtgcttcg 180 gcgctggcga cgatcaaccc gctgaagaccactgttgaag agtcgctgtc gcgtggaatt 240 gctggtagca aaattaaaat tggtactactgcttctactg cgaccgaaac atatgccggc 300 gtcgagccgg atgccaacaa gttgggtgtaattgctgtag caatcgaaga tagtggtgcg 360 ggtgatatta cctttacctt ccagactggtacctctagtc ccaagaatgc tactaaagtt 420 atcactctga accgtactgc ggatggggtctgggcttgta aatctaccca ggatccgatg 480 ttcactccga aaggttctga taac 504 22168 PRT Pseudomonas aeruginosa 22 Ala Leu Glu His His His His His GlyGly Gly Gly Glu Val Ser Ala 1 5 10 15 Leu Glu Lys Glu Val Ser Ala LeuGlu Lys Glu Val Ser Ala Leu Glu 20 25 30 Lys Glu Val Ser Ala Leu Glu LysGlu Val Ser Ala Leu Glu Lys Gly 35 40 45 Gly Gly Gly Glu Phe Ala Arg SerGlu Gly Ala Ser Ala Leu Ala Thr 50 55 60 Ile Asn Pro Leu Lys Thr Thr ValGlu Glu Ser Leu Ser Arg Gly Ile 65 70 75 80 Ala Gly Ser Lys Ile Lys IleGly Thr Thr Ala Ser Thr Ala Thr Glu 85 90 95 Thr Tyr Ala Gly Val Glu ProAsp Ala Asn Lys Leu Gly Val Ile Ala 100 105 110 Val Ala Ile Glu Asp SerGly Ala Gly Asp Ile Thr Phe Thr Phe Gln 115 120 125 Thr Gly Thr Ser SerPro Lys Asn Ala Thr Lys Val Ile Thr Leu Asn 130 135 140 Arg Thr Ala AspGly Val Trp Ala Cys Lys Ser Thr Gln Asp Pro Met 145 150 155 160 Phe ThrPro Lys Gly Ser Asp Asn 165

It is claimed:
 1. A composition for use in treating or preventinginfection by Pseudomonas aeruginosa comprising a P. aeruginosa pilinprotein having an N-terminal peptide region modified to prevent selfassembly of the peptide.
 2. The composition of claim 1, furthercomprising a pharmaceutically acceptable carrier in which the peptide isformulated.
 3. The composition of claim 1, wherein the modifiedN-terminal peptide region lacks an N-terminal portion of native P.aeruginosa.
 4. The composition of claim 3, wherein the modifiedN-terminal region lacks the first 15 up to the first 40 amino acidsresidues of native P. aeruginosa.
 5. The composition of claim 4, whereinthe modified N-terminal region lacks the first 25 up to the first 30amino acids residues of native P. aeruginosa.
 6. The composition ofclaim 1, wherein the N-terminal peptide region is modified to preventalpha-helical formation in the region.
 7. The composition of claim 6,wherein the N-terminal peptide region is modified to contain prolineresidues or strings of glycine residues at positions effective tointerrupt alpha-helical formation.
 8. The composition of claim 1,wherein the N-terminal region of the pilin peptide has been replaced bya peptide moiety capable of forming a coiled-coil homodimer orheterodimer, and the composition contains two modified pilin peptidesjoined through a coiled-coil heterodimer or homodimer interaction. 9.The composition of claim 8, wherein modified pilin peptide has thesequence identified by SEQ ID. NOS. 2, 4, 6, 8 or
 10. 10. Thecomposition of claim 8, wherein the composition is a homodimer orheterodimer containing the modified pilin peptide from two differentPseudomonas strains.
 11. A method of treating or preventing infection byPseudomonas aeruginosa in a subject comprising administering to thesubject, a pharmaceutically effective amount of a P. aeruginosa pilinprotein having an N-terminal peptide region modified to prevent selfassembly of the peptide.
 12. The method of claim 11, wherein the peptideis contained in an aerosolizable vehicle, and said administeringincludes delivering an aerosol of the peptide to the subject's airway.13. The method of claim 11, wherein the modified N-terminal peptideregion lacks an N-terminal portion of native P. aeruginosa.
 14. Themethod of claim 13, wherein the modified N-terminal region lacks thefirst 15 up to the first 40 amino acids residues of native P.aeruginosa.
 15. The method of claim 13, wherein the modified N-terminalregion lacks the first 25 up to the first 30 amino acids residues ofnative P. aeruginosa.
 16. The method of claim 11, wherein the N-terminalpeptide region is modified to prevent alpha-helical formation in theregion.
 17. The method of claim 16, wherein the N-terminal peptideregion is modified to contain proline residues or strings of glycineresidues at positions effective to interrupt alpha-helical formation.18. The method of claim 11, wherein the N-terminal region of the pilinpeptide has been replaced by a peptide moiety capable of forming acoiled-coil homodimer or heterodimer, and the composition contains twomodified pilin peptides joined through a coiled-coil heterodimer orhomodimer interaction.
 19. The method of claim 18, wherein modifiedpilin peptide has the sequence identified by SEQ ID. NOS. 2, 4, 6, 8 or10.